Alanine exchanges of polar amino acids in the transmembrane domains of a platelet-activating factor receptor generate both constitutively active and inactive mutants.

To determine ligand-binding sites of a platelet-activating factor (PAF) receptor, alanine-scanning mutagenesis was carried out. All 23 polar amino acids in the putative 7-transmembrane (TM) domains of a guinea pig PAF receptor were individually replaced with alanine. The ligand-binding properties of mutant receptors were determined after transient expression in COS-7 cells. Mutants in TM II (N58A, D63A), TM III (N100A, T101A, S104A) and TM VII (D289A) displayed higher PAF-binding affinities than seen with the wild-type receptor. In contrast, mutants in TM V (H188A), TM VI (H248A, H249A, Q252A), and TM VII (Q276A, T278A) showed lower affinities. Representative mutants were then stably expressed in Chinese hamster ovary cells to observe PAF-induced cellular signals (arachidonate release, phosphatidylinositol hydrolysis, adenylyl cyclase inhibition). An N100A mutant with the highest affinity was constitutively active and was responsive to lyso-PAF, an inactive derivative of PAF. One nanomolar PAF induced no signals in low affinity mutants, an EC50 value for the wild-type receptor. Three histidines (His-188, His-248, His-249) might form a binding pocket for the phosphate group of PAF, since zinc effectively inhibited ligand binding. Based on these results, a three-dimensional molecular model of PAF and its receptor was generated using bacteriorhodopsin as a reference protein.